Several publications and patent documents are cited in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these citations is incorporated by reference herein.
Hepatitis C virus (HCV) is an enveloped, positive stranded RNA virus that belongs to the Flaviviridae, a family of viruses including human pathogens such as yellow fever virus, dengue virus and West Nile virus (Q. L. Choo et al., Science 244, 359-62 (1989)). Although broad tissue and species tropisms are hallmarks of these viruses, HCV replication has so far only been detected in human and chimpanzee livers. Moreover, for reasons that are not yet understood, HCV RNA levels in infected liver tissue are extremely low, generally below one copy of RNA per cell and hence, can only be detected with PCR, making it difficult to determine whether secondary sites for viral replication exist (J. Boisvert et al., J Infect Dis 184, 827-35 (Oct. 1, 2001); R. E. Lanford, et al., J Virol 69, 8079-83 (1995)).
HCV encodes a polyprotein that is processed proteolytically into ten polypeptides (K. E. Reed, C. M. Rice, Curr Top Microbiol immunol 242, 55-84 (2000)). Three of them are structural proteins required for capsid formation (core) and assembly into enveloped viral particles (E1 and E2). Four of them are enzymes including cysteine and serine proteases (NS2 and NS3), an ATP dependent helicase (NS3) and a RNA-directed RNA polymerase (NS5B). The functions of the remaining three polypeptides, p7, NS4B, and NS5A, for viral replication are not yet known. For study of replication of HCV in tissue culture cells, the structural proteins can be replaced with a selectable marker, such as the neomycin phosphotransferase. See for example FIG. 2, left panel of Lohman et al. (V. Lohmann et al., Science 285, 110-3 (1999)). Replication of such subgenomic HCV replicons in tissue culture cells has so far only been demonstrated in the human hepatoma cell line Huh7, consistent with the narrow host and tissue tropism of HCV infections.
HCV infection poses a significant public health problem. Approximately 3% of the world's population has persistent HCV infection. In 1989, the virus was identified as the major aetiological agent responsible for post-transfusion non-A and non-B hepatitis. Following primary HCV infection, persistent viraemia and chronic hepatitis develop in the majority of cases. Efforts to elucidate the mechanisms behind viral persistence and hepatocellular damage have been frustrated by the lack of a reliable cell culture system for viral propagation in vitro. In addition, as the chimpazee is the only experimental animal susceptible to HCV infection, progress in research is hampered by the lack of a small animal model to facilitate pathophysiological studies as well as the evaluation of antiviral treatment and vaccine strategies.
Furthermore, although the initial HCV infection is asymptomatic, subsequent clinical manifestations of HCV induced liver disease include fibrosis, cirrhosis, and hepatocellular carcinoma (Alter, H. J., and L. B. Seeff. 2000. Semin. Liver Dis. 20:17-35). Combination antiviral therapy with alpha interferon (IFN-α) and ribavirin, a purine nucleoside analogue, arrests disease progression and can lead to sustained recovery in only 45 to 80% of treated patients (Di Bisceglie, A. M., and J. H. Hoofnagle. 2002. Hepatology 36:S121-S127). Additionally, response to IFN-α therapy can vary significantly depending on the viral genotype, ranging from 30 to 40% for genotype 1 to as high as 80% for genotypes 2 and 3. This suggests that viral determinants also play an important role in regulating the cellular IFN response against HCV (Kinzie, J. L., et al., 2001. J. Viral Hepatitis 8:264-269; McHutchison, J. G., et al., 1998. N. Engl. J. Med. 339:1485-1492). The parameters determining the success or failure of antiviral therapy are not understood, and their identification represents a major challenge in HCV biology.
Therefore, there is a desperate need for non-hepatic cell culture systems, and small animal models for the identification and characterization of anti-viral agents for the prevention and treatment of HCV infection. Additionally, there is a need in the art to elucidate the mechanism of HCV inhibition by IFN-α, so that other treatments may be found.